Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome C and activation of caspase-3.
Bcr-Abl expression in leukemic cells is known to exert a potent effect against apoptosis due to antileukemic drugs, but its mechanism has not been elucidated. Recent reports have indicated that a variety of apoptotic stimuli cause the preapoptotic mitochondrial release of cytochrome c (cyt c) into cytosol, which mediates the cleavage and activity of caspase-3 involved in the execution of apoptosis. Whether Bcr-Abl exerts its antiapoptotic effect upstream to the cleavage and activation of caspase-3 or acts downstream by blocking the ensuing degradation of substrates resulting in apoptosis, has been the focus of the present studies. In these, we used (1) the human acute myelogenous leukemia (AML) HL-60 cells that are stably transfected with the bcr-abl gene (HL-60/Bcr-Abl) and express p185 Bcr-Abl; and (2) the chronic myelogenous leukemia (CML)-blast crisis K562 cells, which have endogenous expression of p210 Bcr-Abl. Exposure of the control AML HL-60 cells to high-dose Ara-C (HIDAC), etoposide, or sphingoid bases (including C2 ceramide, sphingosine, or sphinganine) caused the accumulation of cyt c in the cytosol, loss of mitochondrial membrane potential (MMP), and increase in the reactive oxygen species (ROS). These preapoptotic events were associated with the cleavage and activity of caspase-3, resulting in the degradation of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) and DNA fragmentation factor (DFF), internucleosomal DNA fragmentation, and morphologic features of apoptosis. In contrast, in HL-60/Bcr-Abl and K562 cells, these apoptotic stimuli failed to cause the cytosolic accumulation of cyt c and other associated mitochondrial perturbations, as well as the failure to induce the activation of caspase-3 and apoptosis. While the control HL-60 cells showed high levels of Bcl-2 and barely detectable Bcl-xL, HL-60/Bcr-Abl cells expressed high levels of Bcl-xL and undetectable levels of Bcl-2, a pattern of expression similar to the one in K562 cells. Bax and caspase-3 expressions were not significantly different between HL-60/Bcr-Abl or K562 versus HL-60 cells. These findings indicate that Bcr-Abl expression blocks apoptosis due to diverse apoptotic stimuli upstream by preventing the cytosolic accumulation of cyt c and other preapoptotic mitochondrial perturbations, thereby inhibiting the activation of caspase-3 and execution of apoptosis.
Amarante-Mendes
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GP
Division of Hematology/Oncology, Department of Medicine, Winship Cancer Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
Naekyung Kim
C
C
Liu
L
L
Huang
Y
Y
Perkins
C L
CL
Green
D R
DR
Bhalla
K
K
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003561:Cytarabine / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007938:Leukemia / Q000378:metabolism / Q000473:pathology*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D015473:Leukemia, Promyelocytic, Acute; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species / Q000378:metabolism; D013107:Sphingolipids / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C494826:BAX protein, human; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D013107:Sphingolipids; D051028:bcl-2-Associated X Protein; D003561:Cytarabine; D005047:Etoposide; D016044:Fusion Proteins, bcr-abl; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases